This invention relates generally to an apparatus for screening and the pharmacological profiling of compounds modulating a cellular physiological response. This invention also relates to devices for rapid assessment of the properties of compounds that modulate the activities of cells. The compounds investigated may be involved in regulating the activity of signal transduction pathways, cellular responses, cell surface receptors, ion channels, non-selective pores, second messenger pathways, downstream signal transduction pathways, apoptosis, cellular necrosis or any other cellular responses. The devices and methods of the present invention may also be used to perform biochemical analyses, such as Western analyses, Northern analyses, detection of single nucleotide polymorphisms (SNPs), detection of enzymatic activities, or molecular assembly assays.
In some embodiments, this invention relates to methods and apparatus for detecting, evaluating and characterizing the ability and potency of substances to act as agonists or antagonists against receptors and ion channels localized on a cell surface membrane.
Biological cells contain receptor molecules located on their external membrane. The function of these receptors is to xe2x80x9csensexe2x80x9d the cell environment and supply the cell with an input signal about any changes in the environment. In eukaryotic organisms such cell environment is comprised of the neighboring cells and the function of the receptor is to allow cells to communicate with each other directly (the paracrine regulatory system) or indirectly (the endocrine regulatory system) thus achieving harmonized response of a tissue, organ or a whole organism. In prokaryotic cells, the surface localized receptors provide a means for detecting extracellular environment.
Having received such a signal, neurotransmitters, hormones, chemoattractant or chemorepellant substances for example, the surface localized receptors transmit this information about extracellular environment into the cell through specific intracellular pathways in such a way that the cell responds in the specific fashion to accommodate these changes. When there is an altered supply of the external signal molecules or an altered activity of the cell surface molecules, the cell response would be abnormal causing malfunctioning of a tissue or an organ.
In eukaryotic cells, receptor molecules determine the selective response of the cell. Each type of receptor can interact only with a specific set of ligand molecules. For example, adrenergic receptors interact with adrenaline and noradrenaline, cholinergic receptors interact with acetylcholine, serotoninergic receptors interact with 5-hydroxytriptamine, dopamineergic with DOPA and so on. The cells derived from the different tissues invariably express specific sets of tissue receptors. Different types of receptors are connected to different signal transduction pathways. For example, nicotinic cholinergic receptor, upon binding acetylcholine molecule, directly activates sodium channel (Claudio et al., 1987, is incorporated herein by reference). G-protein coupled receptors activate enzymes of second messenger pathways, for example, adenylate cyclase or phospholipase C with subsequent activation of cAMP or phosphoinositide cascades (Divecha and Itvine, 1995, is incorporated herein by reference). Receptor tyrosine kinases activate cascade of MEK/MAPK kinases leading to cell differentiation and proliferation (Marshall, 1995 and Herskowitz, 1995, are incorporated herein by reference)]. Cytokine receptors activate JAK/STAT cascade which in turn can regulate other pathways as well as activate gene transcription (Hill and Treisman, 1995, is incorporated herein by reference).
Together with the receptors, the cell surface meirane carries ion pumps, ion transporters and ion channels. These molecular assemblies work in concert to maintain intracellular ion homeostasis. Any changes in the activity of these systems would cause a shift in the intracellular concentrations of ions and consequently to the cell metabolic response.
Ion pumps act to maintain transmembrane ion gradients utilizing ATP as a source of energy. The examples of the ion pumps are: Na+/K+-ATPase maintaining transmembrane gradient of sodium and potassium ions, Ca2+-ATPase maintaining transmembrane gradient of calcium ions and H+-ATPase maintaining transmembrane gradient of protons.
Ion transporters use the electrochemical energy of transmembrane gradients of one ion species to maintain gradients of other ion counterpart. For example, the Na+/Ca2+-exchanger uses the chemical potential of the sodium gradient directed inward to pump out calcium ions against their chemical potential.
Ion channels, upon activation, allow for the ions to move across the cell membrane in accordance with their electrochemical potential. There are two main types of ion channels: voltage operated and ligand-gated. Voltage operated channels are activated to the open state upon changes in transmembrane electric potential. Sodium channels in the neuronal axon or L-type calcium channels in neuromuscular junctions exemplify this kind of channel. Ligand-gated channels are activated to the open state upon binding a certain ligand with the chemoreceptor part of their molecules. The classical example of ligand-gated channels is nicotinic cholinergic receptor which, at the same time, is the sodium channel.
There are numerous methods for detecting ligand/receptor interaction. The most conventional are methods where the affinity of a receptor to a substance of interest is measured in radioligand binding assays. In these assays, one measures specific binding of a reference radiolabeled ligand molecule in the presence and in the absence of different concentrations of the compound of interest. The characteristic inhibition parameter of the specific binding of the reference radiolabeled ligand with the compound of interest, IC50, is taken as a measure of the affinity of the receptor to this compound (Weiland and Molinoff, 1981 and Swillens et all., 1995, are incorporated herein by reference). Recent advances in microchip sensor technology made it possible to measure direct interactions of a receptor molecule with a compound of interest in real time. This method allows for determination of both association and dissociation rate constants with subsequent calculation of the affinity parameter (F_gerstam et al., 1992, is incorporated herein by reference). While being very precise and convenient, these methods do not allow to distinguish between agonist and antagonist activity of the compound.
The type of biological activity of the compounds, agonist or antagonist, may be determined in the cell based assays. In the methods described in Harpold and Brust, 1995, which is incorporated herein by reference, cells cotransfected with a receptor gene and reporter gene construct, are used to provide means for identification of agonist and antagonist potential pharmaceutical compounds. These methods are inconvenient because they require very laborious manipulations with gene transfection procedures, are highly time consuming and use artificially modified cells. Besides, to prove that the agonistic effect of a particular compound is connected to the stimulation of a transfected receptor, several control experiments with a positive and negative control cell lines should be performed as well.
Most closely related to the methods of this invention are the methods described in Parce et al., 1994, which is incorporated herein by reference. These prior art methods use natural cells and are based on registering the natural cell responses, such as the rate of metabolic acidification, to the biologically active compounds. The disadvantage of the prior art is low throughput speed, each measurement point taking about three minutes. Another disadvantage of the prior art is the use of cells immobilized on the internal surface of the measuring microflow chamber. This leads to the necessity of using separate silicon sensors, or cover slips, with the cells adherent to them for each concentration point of the agonist or antagonist, for the receptors that undergo desensitization upon binding to the agonist molecule. This results in high variability of the experimental results.
Ionized calcium, unlike other intracellular ion events, e.g. changes in the intracellular concentrations of protons, sodium, magnesium, or potassium, serves as the most common element in different signal transduction pathways of the cells ranging from bacteria to specialized neurons (Clapham, 1995, is incorporated herein by reference). There are two major pools which supply signal transduction pathways in the cell with the calcium ions, extracellular space and the endoplasmic reticulum. There are several mechanisms to introduce small bursts of calcium into cytosol for signal transduction.
Both excitable and nonexcitable cells have on their plasma membrane predominantly two receptor classes, G-protein coupled serpentine receptors (GPCSR) and the receptor tyrosine kinases (RTK), that control calcium entry into cell cytoplasm. Both GPCSR and RTK receptors activate phospholipase C to convert phosphatidylinositol into inositol(1,4,5)-trisphosphate (InsP3) and diacylglicerol. InsP3 acts as an intracellular second messenger and activates specialized receptor that spans the endoplasmic reticular membrane. The activation of this receptor triggers release of calcium ions from the endoplasmic reticulum (Berridge, 1993, is incorporated herein by reference). The calcium ions can also enter the cytoplasm of excitable and nonexcitable cell from extracellular environment through specialized voltage-independent Ca2+-selective channels triggered by specific ligands. In nonexcitable cells, hyperpolarization of the plasma cell membrane also enhances entry of calcium ions through passive transmembrane diffusion along the electric potential. For example, opening of potassium channels brings the membrane potential to more negative values inside the cell, thus facilitating Ca2+ entry across the plasma membrane. Excitable cells contain voltage-dependent Ca2+ channels on their plasma membrane, which, upon membrane depolarization, open for a short period of time and allow inflow of Ca2+ from external media into cytoplasm. The endoplasmic reticulum membrane as well as plasma membrane of the excitable cells contains InsP3 receptors and Ca2+-sensitive ryanodine receptors (RyR) releasing Ca2+ from intracellular stores upon membrane receptor triggered phospholipase C activation or depolarization-induced short burst of Ca2+ entry into cell cytoplasm from extracellular media respectively.
It is well established that G-protein coupled serpentine receptors initiate Ca2+ mobilization through the activation of phospholipase Cxcex2 (Sternweis and Smrcka, 1992, is incorporated herein by reference) whereas tyrosine kinase receptors activate phospholipase Cxcex3 with subsequent intracellular Ca2+ mobilization (Berridge and Irvine, 1989, is incorporated herein by reference).
There are many plasma membrane G-protein coupled serpentine receptors, tyrosine kinase growth factor receptors and voltage- and ligand-regulated channels known to initiate intracellular Ca2+ mobilization.
Ca2+ plays an essential role in many functional processes of a cell. For example, Ca2+ affects the cell cycle (Means, 1994, is incorporated herein by reference) and activates specific transcription factors (Sheng et al., 1991, is incorporated herein by reference). Scores of receptors and ion channels use the Ca2+ signal to initiate events as basic as cell motility, contraction, secretion, division etc.
Increases in cytosolic and, consequently, in nuclear concentration of the Ca2+ can also be a cell death promoting signal. For example, prolonged increase in free Ca2+ activates degradation processes in programmed cell death, apoptosis, activates nucleases that cleave DNA and degrade cell chromatin, promotes DNA digestion by direct stimulation of endonucleases, or indirectly by activation of Ca2+-dependent proteases, phosphatases and phospholipases, resulting in a loss of chromatin structural integrity (Nicotera et all., 1994, is incorporated herein by reference).
A development of intracellular fluorescent calcium indicators (Grynkiewicz et all., 1985, is incorporated herein by reference) made it possible for intracellular concentration of free calcium to be measured directly in the living cell. Thus the ability to register changes in intracellular calcium concentration provide the means for monitoring effects of different compounds useful in treating various diseases, whose action is thought to be a result of an interaction with membrane receptors and ion channels.
With the advent of combinatorial chemistry approaches to identify pharmacologically useful compounds, it is increasingly evident that there is a need for methods and apparatuses capable of performing automated characterization of pharmacological profiles and corresponding potencies of the compounds in synthesized combinatorial libraries. This would enable the rapid screening of a large number of compounds in the combinatorial library the identification of those compounds which have biological activity, and the characterization of those compounds in terms of potency, affinity and selectivity.
It is an object of this invention to provide methods for screening and the quantitative characterization of potentially pharmacologically effective compounds that specifically interact with and modulate the activity of cell membrane receptors, ion pumps and ion channels using living cells.
It is an additional object of this invention to provide methods capable of characterizing an affinity of the active compounds to the binding sites of the cell.
It is another additional object of this invention to provide methods to distinguish between agonistic and antagonistic activity of the compounds.
It is yet another additional object of this invention to provide methods to determine the nature of the receptor, ion channel or ion pump entity which is sensitive to the active compounds discovered during the screening process.
It is yet another additional object of this invention to provide methods to characterize cell receptor pattern for particular cell source tissue.
It is yet another additional object of this invention to perform each of the above methods on each member of a series of cell types.
It is yet another additional object of the invention to determine the pattern of cell surface receptors expressed in one or more cell types.
It is yet another additional object of the invention to confirm that a test compound influences the activity of a particular receptor.
It is yet an additional object of the invention to determine the activity of a given receptor in a variety of cell types in which it is expressed.
It is a specific object of this invention to provide an apparatus for fulfillment of the objectives above.
It is yet another additional object of this invention to provide an apparatus for fulfillment of each of the objectives above for each member of a series of cell types.
At least some of these and other objectives are addressed by the various embodiments of the invention disclosed herein.
The present invention addresses the above and other needs by providing a method and corresponding apparatuses which allows the automated characterization of pharmacological profiles and corresponding potencies of compounds in synthesized combinatorial libraries. This enables the rapid screening of a large number of compounds in the combinatorial library, the identification of those compounds which have biological activity, and the characterization of those compounds in terms of potency, affinity and selectivity.
A variety of effects caused by the compounds to be screened may be detected and quantitatively characterized according to the present invention. Preferably, these effects include but are not limited to changes in intracellular concentration of ionized calcium, cAMP or pH, transmembrane potential and other physiological and biochemical characteristics of living cell which can be measured by a variety of conventional means, for example using specific fluorescent, luminescent or color developing dyes.
The present invention also includes methods of screening for agonist or antagonist activity of drugs, methods of characterizing their potency profiles, methods of identifying the receptor expression pattern of cell membrane (xe2x80x9creceptor fingerprintingxe2x80x9d) and methods of determining toxicity profiles for the compounds. In these methods, a steady flow of cells is mixed with flows of the compound and a standard substance. The effects of the compound alone and in mixture with the standard substance are measured and provide the means for pharmacological profiling of the compounds, drug screening and cell receptor pattern characterizing.
In a preferred embodiment of the invention, the compounds to be screened and standard agonist and antagonist substances are organized in a 96-well plate format, or other regular two dimensional array, such as a 48-well and 24-well plate format or an array of test tubes. In another preferred embodiment of the invention, the non-adherent cells are grown in a suspension of freely flowing cells by growing them in an appropriate cell cultivating system.
In another preferred embodiment of the invention, the naturally adherent cells which need attachment to a surface for their growth, are grown in the appropriate cell cultivating system containing commercially available micro spherical beads to which the cells adhere during the growth.
In yet another preferred embodiment of the invention, the naturally adherent cells which need attachment to a surface for their growth, are grown in the cell culture flasks with a subsequent detachment of the cells from the flask bottom with an appropriate detaching reagent.
In accordance with the present invention, either eukaryotic or prokaryotic cells can be used. The cells can be transfected with a gene coding to express a receptor of interest, for example, an orphaned receptor. In addition, the variety of compounds having biologically relevant activity may be used including but not limited to neurotransmitters, hormones, toxins, receptor activators and inhibitors, ion channels and ion pump modulators, irritants and/or drugs.
The cells grown in accordance with the preferred embodiments described above, are mixed with an appropriate fluorescent dye, for example FURA-2AM for measurements concentrations of intracellular calcium or BCECF-AM for measurements of intracellular pH, and are incubated in the appropriate conditions to allow the dye to penetrate into the cell. The cells loaded with a dye are supplied to the apparatus. In the apparatus, the cells are successively mixed with a solutions of the compounds to be tested.
The methods of the present invention may be performed automatically using the apparatti disclosed herein. In particular, the cells or particles may be automatically mixed with test compound(s) and or standard compound(s). The cells or particles may be automatically delivered to the detector. The detector may automatically analyze the cells for particular characteristics, automatically identify test compounds having the desired cellular effects, or automatically identify the presence of a particular molecule in a sample. In some embodiments, a coupling device may automatically deliver the cells or particles to the detector. Calibration solutions may be input automatically. In addition, gradients of test compounds or standard compounds may be automatically provided. The automation of the present methods increases the number of samples which can be evaluated over a given time period.
A first embodiment of the present invention is a method for identifying compounds which produce a cellular response comprising:
(a) using an apparatus to combine a suspension of cells with one or more test compounds to form test mixtures;
(b) directing the test mixtures through a detection zone, the detection zone being capable of detecting a plurality of cellular responses simultaneously; and
(c) simultaneously measuring a plurality of cellular responses to the one or more test compounds in the suspended cells as the test mixtures are flowing through the detection zone.
In one aspect of the first embodiment, the plurality of cellular responses includes a cellular response selected from the group consisting of activation or inhibition of a receptor mediated response, activation or inhibition of an ion channel, activation or inhibition of a non-selective pore, activation or inhibition of a second messenger pathway at a point downstream of a receptor or channel, activation or inhibition of apoptosis, and activation or inhibition of cellular necrosis, and cellular toxicity.
In another aspect of the first embodiment, the plurality of cellular responses are measured by contacting the cells with one or more response indicating reagents which generate signals indicative of particular cellular responses. For example, the signal generated by the response indicating reagents may be fluorescence.
In another aspect of the first embodiment, the step of directing the test mixtures through a detection zone comprises directing the text mixtures through a detection zone which is capable of detecting the cellular responses in individual cells.
In another aspect of the first embodiment, the step of directing the test mixtures through a detection zone comprises directing the test mixtures through a flow cytometer.
In another aspect of the first embodiment, the step of simultaneously measuring a plurality of cellular responses comprises measuring the plurality of cellular responses in the suspended cells as a slug is flowing through the detection zone.
In another aspect of the first embodiment, the suspension of cells comprises more than one type of cell.
A second embodiment of the present invention is a method for identifying compounds which produce a cellular response comprising:
(a) using an apparatus to combine a suspension of cells with one or more test compounds to form test mixtures;
(b) directing the test mixtures through a detection zone, the detection zone being capable of detecting cellular responses in individual cells; and
(c) measuring one or more cellular responses to the one or more test compounds in individual suspended cells as the test mixtures are flowing through the detection zone.
In one aspect of the second embodiment, the one or more cellular responses is selected from the group consisting of activation or inhibition of a receptor mediated response, activation or inhibition of an ion channel, activation or inhibition of a non-selective pore, activation or inhibition of a second messenger pathway at a point downstream of a receptor or channel, activation or inhibition of apoptosis, and activation or inhibition of cellular necrosis, and cellular toxicity.
In another aspect of the second embodiment, wherein the one or more cellular responses is measured by contacting the cells with one or more response indicating reagents which generate signals indicative of particular cellular responses. For example, the signal generated by the response indicating reagents may be fluorescence.
In another aspect of the second embodiment, the step of directing the test mixtures through a detection zone comprises directing the test mixtures through a flow cytometer.
In another aspect of the second embodiment, the suspension of cells comprises more than one type of cell.
In another aspect of the second embodiment, the step of simultaneously measuring a plurality of cellular responses comprises measuring the plurality of cellular responses in the suspended cells as a slug is flowing through the detection zone.
A third embodiment of the present invention is a method of determining whether a sample contains one or more molecules comprising:
using an apparatus to combine one or more agents capable of binding one or more molecules with the sample to form test mixtures;
directing the test mixtures through a detection zone; and
determining whether the one or more agents are bound to the one or more molecules as the one or more agents are flowing through the detection zone.
In one aspect of the third embodiment, the one or more agents are fixed to a solid support. For example, a plurality of agents capable of binding a plurality of molecules may be fixed to uniquely identifiable solid supports. The determining step may comprise detecting a signal from an individual solid support wherein the signal indicates that the agent on the solid support has bound to the molecule.
In another aspect of the third embodiment, the one or more agents are selected from the group consisting of antibodies, nucleic acids, polypeptides, enzymatic substrates, and receptors.
In another aspect of the third embodiment, the one or more molecules are selected from the group consisting of polypeptides, nucleic acids, receptor ligands, enzymatic agonists and enzymatic antagonists.
A fourth embodiment of the present invention is a method of determining whether a sample contains one or more polypeptides comprising:
attaching one or more antibodies which specifically bind the one or more polypeptides to solid supports;
using an apparatus to combine the one or more antibodies attached to solid supports with samples to form test mixtures;
directing the test mixtures through a detection zone; and
determining whether the one or more antibodies are bound to the one or more polypeptides as the solid supports are flowing through the detection zone.
In one aspect of the fourth embodiment, a plurality of antibodies which specifically bind to a plurality of polypeptides are fixed to uniquely identifiable solid supports. The determining step may comprise detecting a signal from an individual solid support wherein the signal indicates that the antibodies have bound to the molecules. The signal may be generated by detectably labeled secondary antibodies which bind the molecules.
A fifth embodiment of the present invention is a method of determining whether a sample contains one or more nucleic acids comprising:
attaching one or more nucleic acid probes which specifically bind the nucleic acids to solid supports;
using an apparatus to combine the one or more probes attached to the solid supports with samples to form test mixtures;
directing the test mixtures through a detection zone; and
determining whether the one or more probes are bound to the one or more nucleic acids as the solid supports are flowing through the detection zone.
In one aspect of the fifth embodiment, a plurality of nucleic acid probes are fixed to uniquely addressable solid supports. The determining step may comprise detecting a signal from an individual solid support wherein the signal indicates that the probes have bound to the nucleic acids. The signal may be generated by detectable nucleic acid probes which bind the nucleic acids.
A sixth embodiment of the present invention is a method of determining whether a sample contains one or more single nucleotide polymorphisms comprising:
performing an extension reaction on a nucleic acid sample using one or more nucleic acid primers having a 3xe2x80x2 end immediately adjacent to the polymorphic base of one or more single nucleotide polymorphisms, thereby incorporating one base into the nucleic acid primers;
attaching the extended primers to solid supports;
directing the solid supports having primers attached thereto through a detection zone in an apparatus; and
determining the identities of the bases incorporated in the extension reaction.
In one aspect of the sixth embodiment, a plurality of primers are fixed to uniquely identifiable solid supports. The determining step comprises detecting a signal from an individual solid support wherein the signal is indicative of the identity of the polymorphic base in a SNP. The signal may be generated by detectably labeled nucleotides incorporated in the extension reaction.
In another aspect, the first embodiment further comprises the steps of:
(d) combining a suspension of the cells with one or more standard compounds having a known effect on the cellular response of the cells to form standard mixtures;
(e) directing the standard mixtures through the detection zone; and
(f) measuring the cellular response of the cells to the one or more standard compounds.
In the above aspect of the first embodiment, the one or more standard compounds and the one or more test compounds may be simultaneously mixed with the cells in the combining steps, and the measuring step detects the known effect or an alteration of the known effect. The one or more standard compounds is an antagonist of the cellular response. The one or more standard compounds is an agonist of the cellular response. The suspension of cells may comprise more than one cell type. The step of directing the test mixtures through a detection zone may comprise directing the test mixtures through a flow cytometer.
A seventh embodiment of the present invention is a method of obtaining cells having a desired phenotype or cellular response profile comprising the steps of:
(a) using an apparatus to combine a suspension of cells with one or more compounds which produce a cellular response to form test mixtures;
(b) directing the test mixtures through a detection zone, the detection zone being capable of detecting a plurality of cellular parameters simultaneously;
(c) simultaneously measuring a plurality of cellular parameters in the suspended cells as the test mixtures are flowing through the detection zone; and
(d) delivering cells having a desired phenotype or cellular response profile into a receptacle.
An eighth embodiment of the present invention is a method of obtaining cells having a desired phenotype or cellular response profile comprising the steps of:
(a) using an apparatus to combine a suspension of cells with one or more compounds which produce a cellular response to form test mixtures;
(b) directing the test mixtures through a detection zone, the detection zone being capable of detecting cellular responses in individual cells;
(c) simultaneously measuring a plurality of cellular parameters in the suspended cells as the test mixtures are flowing through the detection zone; and
(d) delivering cells having a desired phenotype or cellular response profile into a receptacle.
A ninth embodiment of the present invention is an apparatus comprising:
a test compound source;
a test substrate source;
a mixing chamber in fluid communication with said test compound source and said test substrate source, wherein said mixing chamber is adapted for combining a test compound received from said test compound source with a test substrate received from said test substrate source to generate a mixture; and
a detector in fluid communication with said mixing chamber, said detector being capable of detecting an interaction between said test compound and said test substrate while said mixture is passing through said detector.
A tenth embodiment of the present invention is an apparatus comprising:
one or more sample inputs for sequentially providing multiple samples, the samples comprising one or more test compounds to be evaluated for the ability to produce a cellular response or a solution to be evaluated for the presence of molecules;
one or more cell or particle inputs for providing a cell suspension or particles;
a mixing zone, coupled to the sample input, for receiving the samples, receiving the cell suspension or particles from the cell or particle input and mixing each sample with the cell suspension or particles; and
a detector capable of detecting a plurality of signals simultaneously, the detector being coupled to the mixing zone and being capable of simultaneously measuring a plurality of cellular responses in the suspended cells or simultaneously determining whether a plurality of molecules are present in the samples.
In one aspect of the tenth embodiment, the detector detects a signal from a single cell in the cell suspension or from a single particle.
In another aspect of the tenth embodiment, the detector is a flow cytometer.
In another aspect of the tenth embodiment, the apparatus further comprises a coupler disposed between the mixing zone and the detector. The coupler may deliver slugs comprising the samples and the cell suspension or particles to the detector. In some embodiments the coupler delivers substantially undiluted slugs to the detector.
In another aspect of the tenth embodiment, the detector delivers cells having a desired phenotype or cellular response to a receptacle.
In another aspect of the tenth embodiment, the sample input is an automated robotic sampler capable of selecting a specified test compound from a library of test compounds. The device may further comprise a controller, coupled to the sample input, for controlling the operation of the test compound sampler; and
a computer, coupled to the controller, for sending command signals to the controller in accordance with a software program implemented by the computer, thereby controlling the selection and retrieval of test compounds by the sample input from the test compound library. The apparatus may further comprise a gradient pump having an input and an output, coupled to the sample input, for adjusting the concentration level of the test compound transferred to the mixing zone from the sample input, wherein:
the sample input comprises:
a first intake nozzle for receiving the specified test compound;
a second intake nozzle for receiving a buffer solution; and
wherein the gradient pump is coupled to the first and second intake nozzles and receives specified concentrations of the test compound by adjusting the amount of test compound and buffer solution received by the first and second intake nozzles, respectively, wherein the buffer solution is a diluting agent of the test compound. The apparatus may further comprise a second pump, coupled to an input of the mixing zone, for pumping the suspension of cells or the particles from the cell or particle input into the mixing zone. The apparatus may further comprise a reaction developing line, having an input coupled to an output of the mixing zone and an output in fluid communication with the detector wherein the reaction developing line provides a reaction time delay for a mixture received from the mixing zone. The apparatus may further comprise:
a pump, coupled to the output of the detector, for providing negative pressure to the apparatus;
a proportionating valve, coupled to the sample input, for adjusting the concentration level of the test compound transferred to the mixing zone from the sample input, wherein the sample input further comprises:
a first intake nozzle for receiving the specified test compound;
a second intake nozzle for receiving a buffer solution; and
the proportionating valve receives specified concentrations of the test compound by adjusting the amount of test compound and buffer solution received by the first and second intake nozzles, respectively, wherein the buffer solution is a diluting agent of the test compound.
In another aspect of the tenth embodiment, the apparatus further comprises a plurality of cell suspension reservoirs.
In another aspect of the tenth embodiment, the apparatus further comprises a standard compound sampler, coupled to the mixing zone, for providing one or more standard compounds having a known effect on the cellular response of the suspended cells or a known interaction with the particles, wherein the mixing zone receives the one or more standard compounds from the standard compound sampler and mixes the one or more standard compounds with the suspended cells or particles and the detector measures the cellular response of the suspended cells to the one or more standard compounds or the interaction between the one or more standard compounds and the particles. The mixing zone may simultaneously mix the sample and the one or more standard compounds with the suspended cells or the particles and the detector detects the known effect or an alteration of the known effect on the cellular responses of the suspended cells or the known interaction between the standard compound and the particles or an alteration of the known interaction between the standard compound and the particles. The standard compound sampler may be an automated robotic sampler capable of selecting a specified standard compound from a library of standard compounds.
A elventh embodiment of the present invention is an apparatus comprising:
a sample input for sequentially providing multiple samples, the samples comprising one or more test compounds to be evaluated for the ability to produce a cellular response or a solution to be evaluated for the presence of molecules;
a cell or particle input for providing a cell suspension or particles;
a mixing zone, coupled to the sample input, for receiving the samples, receiving the cell suspension or particles from the cell or particle input and mixing each sample with the cell suspension or particles; and
a detector which detects one or more signals from a single cell or particle, the detector being coupled to the mixing zone and being capable of measuring one or more cellular responses in the suspended cells or determining whether one or more molecules are present in the samples.
In one aspect of the elventh embodiment, the apparatus further comprises a coupler disposed between the mixing zone and the detector. The coupler may deliver slugs comprising the samples and the cell suspension or particles to the detector. The coupler may deliver substantially undiluted slugs to the detector. The apparatus may further comprise an input system for inputting solutions into the mixing zone. The detector may deliver cells having a desired phenotype or cellular response to a receptacle.
An twelfth embodiment of the present invention is an apparatus comprising:
a sample input for sequentially providing multiple samples, the samples comprising one or more test compounds to be evaluated for the ability to produce a cellular response or a solution to be evaluated for the presence of molecules;
a cell or particle input for providing a cell suspension or particles;
a mixing zone, coupled to the sample input, for receiving the samples, receiving the cell suspension or particles from the cell or particle input and mixing each sample with the cell suspension or particles; and
a detector capable of detecting a plurality of signals simultaneously, the detector being coupled to the mixing zone and being capable of simultaneously measuring a plurality of cellular responses in the suspended cells or simultaneously determining whether a plurality of molecules are present in the samples.
In one aspect of the twelfth embodiment, the detector detects a signal from a single cell in the cell suspension or from a single particle.
In another aspect of the twelfth embodiment, the detector is a flow cytometer.
In another aspect of the twelfth embodiment, the apparatus further comprises a coupler disposed between the mixing zone and the detector. The coupler may deliver slugs comprising the samples and the cell suspension or particles to the detector.
The coupler may deliver substantially undiluted slugs to the detector.
In another aspect of the twelfth embodiment, the apparatus further comprises an input system for inputting solutions into the mixing zone.
In another aspect of the twelfth embodiment, the detector delivers cells having a desired phenotype or cellular response to a receptacle.
In another aspect of the twelfth embodiment, the apparatus further comprises a standard compound sampler, coupled to the mixing zone, for providing one or more standard compounds having a known effect on the cellular response of the suspended cells or a known interaction with the particles, wherein the mixing zone receives the one or more standard compounds from the standard compound sampler and mixes the one or more standard compounds with the suspended cells or particles and the detector measures the cellular response of the suspended cells to the one or more standard compounds or the interaction between the one or more standard compounds and the particles. The mixing zone may simultaneously mix the sample and the one or more standard compounds with the suspended cells or the particles and the detector detects the known effect or an alteration of the known effect on the cellular responses of the suspended cells or the known interaction between the standard compound and the particles or an alteration of the known interaction between the standard compound and the particles.
The apparatus may further comprise:
a first gradient device, coupled to the sample input, for automatically adjusting the concentration level of the one or more test compounds transferred to the mixing zone from the sample input; and
a second gradient device, coupled to the standard compound sampler, for automatically adjusting the concentration level of the one or more standard compounds transferred to the mixing zone from the standard compound sampler. The apparatus may further comprise a switching valve, coupled to the first and second gradient devices at an input of the switching valve and coupled to the mixing zone at an output of the switching valve, for selectively switching the flow of a concentration of the one or more test compounds or a concentration of the one or more standard compounds or both to the mixing zone where the one or more test compounds and/or the one or more standard compounds are then mixed with the suspension of cells or the particles. The apparatus may further comprise a calibration unit, coupled to the switching valve, wherein the switching valve also selectively switches the flow of a calibration solution provided by the calibration unit into the mixing zone where the calibration solution is mixed with the suspension of cells or the particles. The apparatus may further comprise reaction developing lines coupled to the output of the mixing zone, for receiving a mixture of the cell suspension or the particles mixed with either the one or more test compounds, the one or more standard compounds or the calibration solution, and providing a flow path for the mixture such that there is adequate time for the suspended cells or particles to react with the one or more test compounds, the one or more standard compounds or the calibration solution, wherein the reaction developing lines are further coupled to the input of the detector which receives the mixture from the reaction developing lines.
In another aspect of the twelfth embodiment, the detector simultaneously detects a plurality of cellular responses including a cellular response selected from the group consisting of activation or inhibition of a receptor mediated response, activation or inhibition of an ion channel, activation or inhibition of a non-selective pore, activation or inhibition of a second messenger pathway at a point downstream of a receptor or channel, activation or inhibition of apoptosis, activation or inhibition of cellular necrosis, and cellular toxicity
The apparatus may further comprise:
a controller, coupled to the first and second gradient devices, the sample input, the standard compound sampler, the switching valve, and the coupler for controlling their operation; and
a computer, coupled to the controller, for sending command signals to the controller in accordance with a software program implemented by the computer, wherein the computer is also coupled to the detector in order to send and receive signals indicative of a cellular response or the presence of a molecule in the sample to and from the detector.
In another aspect of the twelfth embodiment, the sample input is an automated robotic sampler capable of selecting a specified test compound from a library of test compounds. The apparatus may further comprise:
a controller, coupled to the sample input, for controlling the operation of the test compound sampler; and
a computer, coupled to the controller, for sending command signals to the controller in accordance with a software program implemented by the computer, thereby controlling the selection and retrieval of test compounds by the sample input from the test compound library.
The apparatus may further comprise a gradient pump having an input and an output, coupled to the sample input, for adjusting the concentration level of the test compound transferred to the mixing zone from the sample input, wherein:
the sample input comprises:
a first intake nozzle for receiving the specified test compound;
a second intake nozzle for receiving a buffer solution; and
wherein the gradient pump is coupled to the first and second intake nozzles and receives specified concentrations of the test compound by adjusting the amount of test compound and buffer solution received by the first and second intake nozzles, respectively, wherein the buffer solution is a diluting agent of the test compound. The apparatus may further comprise a standard compound sampler for providing a sample of a standard compound to the mixing zone.
The standard compound sampler may be an automated robotic sampler capable of selecting a specified standard compound from a library of standard compounds. The apparatus may further comprise a second gradient pump having an input and an output, coupled to the standard compound sampler, for adjusting the concentration level of the standard compound provided to the mixing zone from standard compound sampler, wherein:
the standard compound sampler comprises:
a third intake nozzle for receiving the specified standard compound;
a fourth intake nozzle for receiving a buffer solution; and
wherein the second gradient pump is coupled to the third and fourth intake nozzles and receives specified concentrations of the standard compound by adjusting the amount of standard compound and buffer solution received by the third and fourth intake nozzles, respectively, wherein the buffer solution is a diluting agent of the standard compound. The apparatus may further comprise a second mixing zone coupled to the outputs of the first and second gradient pumps, for receiving and mixing the specified concentrations of the specified test compound and the specified standard compound, such that the output of the second mixing zone is provided to the first mixing zone. The apparatus may further comprise:
a calibration unit for providing a calibration solution; and
a switching valve, having a first input coupled to the second mixing zone, a second input coupled to the calibration unit, and an output coupled to the first mixing zone, for switching between the flow of either a compound mixture from the second mixing zone or the calibration solution from the calibration unit and then providing the flow to the first mixing zone where it may be mixed with the cell suspension or particles. The calibration unit may comprise:
a calibration maximum solution which provides for maximal cell response or maximum interaction of particles with a molecule when mixed with the cell suspension or particles;
a calibration minimum solution which provides for minimal cell response or minimal interaction of a particle with a molecule when mixed with the cell suspension or particles;
a diverting valve having a first input coupled to the calibration maximum solution and a second input coupled to the calibration minimum solution, for switching between the flow of either the calibration maximum solution or calibration minimum solution; and
a pump, coupled to the output of the diverting valve and an input of the switching valve, for pumping either the calibration maximum or calibration minimum solution from the diverting valve into the switching valve. The apparatus may further comprise a second pump, coupled to an input of the first mixing zone, for pumping the suspension of cells or the particles from the cell or particle input into the first mixing zone. The apparatus may further comprise a reaction developing line, having an input coupled to an output of the first mixing zone and an output coupled to an input of the detector, for providing a flow path and a reaction time delay for a mixture received from the first mixing zone and for providing the mixture to the detector. The apparatus may further comprise:
a controller, coupled to the first and second gradient pumps, the sample input, the standard compound sampler and the switching valve, the first and second mixing zones, the first and second pumps and the diverting valve for controlling their operation; and
a computer, coupled to the controller, for sending command signals to the controller in accordance with a software program implemented by the computer, wherein the computer is also coupled to the detector in order to send and receive signals indicative of a cellular response or the presence of a molecule in a sample to and from the detector. The detector may detect a plurality of cellular responses including a cellular response selected from the group consisting of activation or inhibition of a receptor mediated response, activation or inhibition of an ion channel, activation or inhibition of a non-selective pore, activation or inhibition of a second messenger pathway at a point downstream of a receptor or channel, activation or inhibition of apoptosis, activation or inhibition of cellular necrosis, and cellular toxicity.
The apparatus may further comprise:
a pump, coupled to the output of the detector, for providing negative pressure to the apparatus;
a proportionating valve, coupled to the sample input, for adjusting the concentration level of the test compound transferred to the mixing zone from the sample input, wherein the sample input further comprises:
a first intake nozzle for receiving the specified test compound;
a second intake nozzle for receiving a buffer solution; and
the proportionating valve receives specified concentrations of the test compound by adjusting the amount of test compound and buffer solution received by the first and second intake nozzles, respectively, wherein the buffer solution is a diluting agent of the test compound. The apparatus may further comprise:
an automated standard compound sampler capable of selecting a specified standard compound from a library of standard compounds, the standard compound sampler including a third intake nozzle for receiving the specified standard compound and a fourth intake nozzle for receiving a buffer solution; and
a second proportionating valve, coupled to the third and fourth intake nozzles, for receiving specified concentrations of the standard compound by adjusting the amount of standard compound and buffer solution received by the third and fourth intake nozzles, respectively, wherein the buffer solution is a diluting agent of the standard compound. The apparatus may further comprise:
a first priming valve, coupled to the output of the first proportionating valve, for receiving the specified concentration of the test compound and providing the test compound to the mixing zone;
a second priming valve, coupled to the output of the second proportionating valve, for receiving the specified concentration of the standard compound and providing the standard compound to the mixing zone. The apparatus may further comprise:
a calibration unit including a calibration maximum solution which provides for maximal cell response or maximum interaction with particles when mixed with the cell suspension or particles and a calibration minimum solution which provides for minimal cell response or minimal interaction with particles when mixed with the cell suspension;
a first diverting valve, having a first input coupled to the calibration maximum solution and a second input coupled to the calibration minimum solution, for switching between the flow of either the calibration maximum solution or calibration minimum solution;
a second diverting valve, having a first input coupled to the output of the mixing zone and a second input coupled to the output of the first diverting valve, for switching between the flow of either a calibration solution from the first diverting valve or a mixture from the mixing zone;
a third priming valve, coupled to the output of second diverting valve, for receiving a mixture from the second diverting valve; and
a second mixing zone, coupled to the output of the third priming valve, for mixing a mixture provided by the third priming valve with the cell suspension or particles, wherein the cell or particle input and the detector are coupled to the second mixing zone instead of the first mixing zone. The apparatus may further comprise a reaction developing line, having an input coupled to the output of the second mixing zone and an output coupled to an input of the detector, for providing a flow path and a reaction time delay for a mixture received from the second mixing zone before the mixture reaches the detector. The cell or particle input may comprise:
a cell suspension or particle reservoir;
a buffer reservoir;
a third diverting valve, having a first input coupled to the cell suspension or particle reservoir and a second input coupled to the buffer reservoir, for adjusting the concentration of the cell suspension or the particles, wherein the buffer is a diluting agent of the cell suspension or particles; and
a fourth priming valve, coupled to the output of the third diverting valve, for receiving the cell suspension or particle mixture from the third diverting valve and providing this mixture to the second mixing zone. The detector may detect a plurality of cellular responses including a cellular response selected from the group consisting of activation or inhibition of a receptor mediated response, activation or inhibition of an ion channel, activation or inhibition of a non-selective pore, activation or inhibition of a second messenger pathway at a point downstream of a receptor or channel, activation or inhibition of apoptosis, activation or inhibition of cellular necrosis, and cellular toxicity. The apparatus may further comprise a plurality of cell suspension reservoirs.
A thirteenth embodiment of the present invention is an apparatus comprising:
a sample input for sequentially providing multiple samples, the samples comprising one or more test compounds to be evaluated for the ability to produce a cellular response or a solution to be evaluated for the presence of molecules;
a cell or particle input for providing a cell suspension or particles;
a mixing zone, coupled to the sample input, for receiving the samples, receiving the cell suspension or particles from the cell or particle input and mixing each sample with the cell suspension or particles; and
a detector which detects one or more signals from a single cell or particle, the detector being coupled to the mixing zone and being capable of measuring one or more cellular responses in the suspended cells or determining whether one or more molecules are present in the samples.
In one aspect of the thirteenth embodiment, the detector is a flow cytometer.
In another aspect of the thirteenth embodiment, the detector delivers cells having a desired phenotype or cellular response to a receptacle.
In another aspect of the thirteenth embodiment, further comprising a coupler disposed between the mixing zone and the detector. The coupler may deliver slugs comprising the samples and the cell suspension or particles to the detector. The coupler may deliver substantially undiluted slugs to the detector.
In another aspect of the thirteenth embodiment, the apparatus further comprises a standard compound sampler, coupled to the mixing zone, for providing a sample of one or more standard compounds having a known effect on the cellular response of the suspended cells or a known interaction with the particles, wherein the mixing zone receives the sample of the one or more standard compounds from the standard compound sampler and mixes the one or more standard compounds with the suspended cells or particles and the detector measures the cellular response of the suspended cells to the one or more standard compounds or the interaction between the one or more standard compounds and the particles. The mixing zone may simultaneously mix the one or more test compounds and the one or more standard compounds with the suspended cells or the particles and the detector detects the known effect or an alteration of the known effect on the cellular responses of the suspended cells or the known interaction between the one or more standard compounds and the particles or an alteration of the known interaction between the one or more standard compounds and the particles. The apparatus may further comprise:
a first gradient device, coupled to the sample input, for automatically adjusting the concentration level of the one or more test compounds transferred to the mixing zone from the sample input; and
a second gradient device, coupled to the stand compound sampler, for automatically adjusting the concentration level of the one or more standard compounds transferred to the mixing zone from the standard compound sampler. The apparatus may further comprise a switching valve, coupled to the first and second gradient devices at an input of the switching valve and coupled to the mixing zone at an output of the switching valve, for selectively switching the flow of a concentration of the one or more test compounds or a concentration of the standard compound or both to the mixing zone where the one or more test compounds and/or the one or more standard compounds are then mixed with the suspension of cells or the particles. The apparatus may further comprise a calibration unit, coupled to the switching valve, wherein the switching valve also selectively switches the flow of a calibration solution provided by the calibration unit into the mixing zone where the calibration solution is mixed with the suspension of cells or the particles. The apparatus may further comprise reaction developing lines coupled to the output of the mixing zone, for receiving a mixture of the cell suspension or the particles mixed with either the one or more test compounds, the one or more standard compounds or the calibration solution, and providing a flow path for the mixture such that there is adequate time for the suspended cells or particles to react with the one or more test compounds, the standard compound or the calibration solution, wherein the reaction developing lines is further coupled to the input of the detector which receives the mixture from the reaction developing lines. The detector may detect one or more cellular responses selected from the group consisting of are selected from the group consisting of activation or inhibition of a receptor mediated response, activation or inhibition of an ion channel, activation or inhibition of a non-selective pore, activation or inhibition of a second messenger pathway at a point downstream of a receptor or channel, activation or inhibition of apoptosis, activation or inhibition of cellular necrosis, and cellular toxicity. The apparatus may further comprise:
a controller, coupled to the first and second gradient devices, the sample input, the standard compound sampler, the switching valve, and the coupler for controlling their operation; and
a computer, coupled to the controller, for sending command signals to the controller in accordance with a software program implemented by the computer, wherein the computer is also coupled to the detector in order to send and receive signals indicative of a cellular response or the presence of a molecule in the sample to and from the detector.
In another aspect of the thirteenth embodiment, the sample input is an automated robotic sampler capable of selecting a specified test compound from a library of test compounds. The apparatus may further comprise:
a controller, coupled to the sample input, for controlling the operation of the test compound sampler; and
a computer, coupled to the controller, for sending command signals to the controller in accordance with a software program implemented by the computer, thereby controlling the selection and retrieval of test compounds by the sample input from the test compound library. The apparatus may further comprise a gradient pump having an input and an output, coupled to the sample input, for adjusting the concentration level of the test compound transferred to the mixing zone from the sample input, wherein:
the sample input comprises:
a first intake nozzle for receiving the specified test compound;
a second intake nozzle for receiving a buffer solution; and
wherein the gradient pump is coupled to the first and second intake nozzles and receives specified concentrations of the test compound by adjusting the amount of test compound and buffer solution received by the first and second intake nozzles, respectively, wherein the buffer solution is a diluting agent of the test compound.
The apparatus may further comprise:
a pump, coupled to the output of the detector, for providing negative pressure to the apparatus;
a proportionating valve, coupled to the sample input, for adjusting the concentration level of the test compound transferred to the mixing zone from the sample input, wherein the sample input further comprises:
a first intake nozzle for receiving the specified test compound;
a second intake nozzle for receiving a buffer solution; and
the proportionating valve receives specified concentrations of the test compound by adjusting the amount of test compound and buffer solution received by the first and second intake nozzles, respectively, wherein the buffer solution is a diluting agent of the test compound. The apparatus may further comprise:
an automated standard compound sampler capable of selecting a specified standard compound from a library of standard compounds, the standard compound sampler including a third intake nozzle for receiving the specified standard compound and a fourth intake nozzle for receiving a buffer solution; and
a second proportionating valve, coupled to the third and fourth intake nozzles, for receiving specified concentrations of the standard compound by adjusting the amount of standard compound and buffer solution received by the third and fourth intake nozzles, respectively, wherein the buffer solution is a diluting agent of the standard compound. The apparatus may further comprise:
a first priming valve, coupled to the output of the first proportionating valve, for receiving the specified concentration of the test compound and providing the test compound to the mixing zone;
a second priming valve, coupled to the output of the second proportionating valve, for receiving the specified concentration of the standard compound and providing the standard compound to the mixing zone. The apparatus may further comprise:
a calibration unit including a calibration maximum solution which provides for maximal cell response or maximum interaction with particles when mixed with the cell suspension or particles and a calibration minimum solution which provides for minimal cell response or minimal interaction with particles when mixed with the cell suspension;
a first diverting valve, having a first input coupled to the calibration maximum solution and a second input coupled to the calibration minimum solution, for switching between the flow of either the calibration maximum solution or calibration minimum solution;
a second diverting valve, having a first input coupled to the output of the mixing zone and a second input coupled to the output of the first diverting valve, for switching between the flow of either a calibration solution from the first diverting valve or a mixture from the mixing zone;
a third priming valve, coupled to the output of the second diverting valve, for receiving a mixture from the second diverting valve; and
a second mixing zone, coupled to the output of the third priming valve, for mixing a mixture provided by the third priming valve with the cell suspension or particles, wherein the cell or particle input and the detector are coupled to the second mixing zone instead of the first mixing zone. The apparatus may further comprise a reaction developing line, having an input coupled to the output of the second mixing zone and an output coupled to an input of the detector, for providing a flow path and a reaction time delay for a mixture received from the second mixing zone before the mixture reaches the detector. The cell or particle input may comprise:
a cell suspension or particle reservoir;
a buffer reservoir;
a third diverting valve, having a first input coupled to the cell suspension or particle reservoir and a second input coupled to the buffer reservoir, for adjusting the concentration of the cell suspension or the particles, wherein the buffer is a diluting agent of the cell suspension or particles; and
a fourth priming valve, coupled to the output of the third diverting valve, for receiving the cell suspension or particle mixture from the third diverting valve and providing this mixture to the second mixing zone. The detector may detect a plurality of cellular responses including a cellular response selected from the group consisting of activation or inhibition of a receptor mediated response, activation or inhibition of an ion channel, activation or inhibition of a non-selective pore, activation or inhibition of a second messenger pathway at a point downstream of a receptor or channel, activation or inhibition of apoptosis, activation or inhibition of cellular necrosis, and cellular toxicity. The apparatus may further comprise a plurality of cell suspension reservoirs.
In another aspect, the invention relates to a method for determining the effect of each of a plurality of test agents on cells from a subject comprising:
(a) obtaining cells from the subject;
(b) combining each of a plurality of samples comprising the cells with one or more of the test agents to form each of a plurality of test mixtures; and
(c) sequentially directing each of said plurality of test mixtures through a detection zone in an apparatus, said detection zone being capable of detecting the effect of said agents on said cells.
In an embodiment, the above method further comprises determining whether said one or more test agents has a desired effect on said cells from said subject.
In another embodiment, the above method further comprises using an automated and computer controlled apparatus to sequentially combine each of a plurality of samples comprising said cells with one or more said agents.
In a further embodiment, the agents in the above method are selected from the group consisting of chemical compounds, biological molecules, and test cells. The agents may be chemical compounds.
In an embodiment, the subject in the above method is a mammal. The mammal may be selected from the group consisting of mouse, rat, rabbit, dog, cat, sheep, goat, cattle, pig, monkey, and human. In other embodiments, the mammal is a human.
In another embodiment, the cells are normal cells. The normal cells may be selected from the group consisting of cells involved in generating or modulating an immune system.
In another embodiment, however, the cells are abnormal cells. The abnormal cells are selected from the group consisting of cancer cells.
In yet another embodiment, the test agents are selected from the group consisting of agents for treating cancer, immunosuppressive drugs, antibiotics, anti-inflammatory drugs, neurotransmitters, growth hormones, and analgesics.
An embodiment of the invention provides for the test mixtures of the above method to further comprise one or more response indicating agents. The response indicating agents may indicate a decrease or cessation of replication, or they may indicate cell death.
In another embodiment, the plurality of test mixtures vary according to a condition selected from the group consisting of the concentration of said one or more test agents, incubation condition, type of cell, type of test agent, and number of test agents, or a combination thereof.
In further embodiments, the detection zone is capable of detecting a plurality of cellular responses simultaneously.
In an embodiment, the determining step comprises simultaneously measuring a plurality of said extent of response of said cells to said one or more test compounds as each of said plurality of test mixtures are flowing through said detection zone.
In another embodiment, the detection zone is capable of detecting a cellular response as a single cell is flowing through said detection zone. The detection zone may comprise a flow cytometer.
In another of the invention, the above method further comprises conducting a genetic analysis to determine whether said subject is likely to have a desirable or adverse response to said one or more test agents. The genetic analysis may comprise determining whether said subject has an allele of one or more single nucleotide polymorphisms which indicates that said subject will have said desirable or adverse response.
Another embodiment of the invention provides for the above method further comprising conducting a cellular analysis to determine whether said subject is likely to have a desirable or adverse response to said one or more test compounds. The above cellular analysis may comprise determining whether said one or more test compounds will be maintained at effective concentrations in said cells. Further, the above cellular analysis may comprise measuring the level of activity of one or more multidrug resistance transporters in said cells.
In another embodiment, the test agent of the above methods comprises test cells. Within this embodiment, the subject may be a mammal. The mammal may be selected from the group consisting of mouse, rat, rabbit, dog, cat, sheep, goat, cattle, pig, monkey, and human.
In another embodiment, the cells are normal cells. These normal cells may be cells involved in generating or modulating an immune response.
In yet another embodiment, the cells are abnormal cells. These abnormal cells may be cancer cells.
In an embodiment, the test agents are selected from the group consisting of agents for treating cancer, immunosuppressive drugs, antibiotics, anti-inflammatory drugs, neurotransmitters, growth hormones, and analgesics.
In another embodiment, the test mixtures further comprise one or more response indicating agents. The response indicating agents may indicate a decrease or cessation of replication. The response indicating agents may instead indicate cell death.
In an embodiment, the plurality of test mixtures vary according to a condition selected from the group consisting of the concentration of said one or more test agents, incubation condition, type of cell, type of test agent, and number of test agents, or a combination thereof.
In a further embodiment, the detection zone is capable of detecting a plurality of cellular responses simultaneously.
In still another embodiment, the determining step comprises simultaneously measuring a plurality of said extent of response of said cells to said one or more test compounds as each of said plurality of test mixtures are flowing through said detection zone.
In yet other embodiments, the detection zone is capable of detecting a cellular response as a single cell is flowing through said detection zone. The detection zone may comprise a flow cytometer.
In another embodiment, the above method further comprises conducting a genetic analysis to determine whether said subject is likely to have a desirable or adverse response to said one or more test agents. Another embodiment relates to the above method further comprising conducting a genetic analysis to determine whether said subject is likely to have a desirable or adverse response to said test cells. The genetic analysis may comprise determining whether said subject has an allele of one or more single nucleotide polymorphisms which indicates that said subject will have said desirable or adverse response.
In an embodiment, the above method further comprising conducting a cellular analysis to determine whether said subject is likely to have a desirable or undesirable response to said test cells. The cellular analysis may comprise determining whether said test cells express one or molecules on their surface which will cause a desirable or undesirable response. The one or molecules may comprise major histocompatibility molecules.
In yet another embodiment, the test agents are test cells from a candidate organ or tissue to be tested for use in an organ or tissue transplant.
Further details on the latter aspects of the invention are set forth in Example 22.